KR101241551B1 - Refolding transforming growth factor beta family proteins - Google Patents

Abstract

Compositions and methods for folding proteins belonging to the transforming growth factor beta superfamily are disclosed. The compositions and methods allow for the folding of such proteins when produced in an expression system that does not yield a properly folded, biologically active product.

Description

REFOLDING TRANSFORMING GROWTH FACTOR BETA FAMILY PROTEINS}

This application claims priority to Provisional Application No. 60 / 602,825, filed August 19, 2004. The entire contents of the application are hereby incorporated by reference.

The present invention relates to compositions and methods for refolding proteins belonging to the transforming growth factor beta superfamily.

New blastine, known as artemin and enovin, is a 24-kDa homodimeric secreted protein that promotes the survival of peripheral and central nervous system neurons such as dopaminergic neurons (Baudet et al., 2000, Development , 127: 4335). Roseblad et al., 2000, Mol . Cell Neurosci. , 15 (2): l99; GenBank (TM) AF120274). The gene encoding Nublastin was cloned and the sequence identified (Roseblad et al., 2000, Mol . Cell Neurosci ., 15 (2): 199; Baloh et al., Neuron , 21: 1291).

Nublastine is a member of the glioma cell line-derived neuronal growth factor (GDNF) ligand family. At the cellular level, GDNF members activate the receptor tyrosine kinase, RET. RET is associated with the glycosylphosphatidyl inositol (GPI) bound membrane protein GDNF family receptor α (GFRα), a co-receptor that provides ligand specificity for RET. Four GFRα are known (GFRα1-4). Nublastin is a ternary signal complex (Baudet et al., 2000, Development , 127: predominantly localized in nociceptive sensory neurons (Orozco et al., 2001, Eur. J. Neurosci ., 13 (11): 2177)). 4335; Baloh et al., 1998, Neuron , 21: 1291) and binds to GFRα3 with RET. These neurons detect pain and damage. Thus, nublastin has clinical application in the general treatment of neuropathy, more particularly in the treatment of neuropathic pain.

Nublastin and other GDNF family members are members of the transforming growth factor beta (TGF beta) superfamily and are thus characterized by the presence of seven conserved cysteine residues with similar intervals forming the structure of the cysteine knot. (Saarma, 1999, Microsc . Res. Tech. , 45: 292). Each monomer comprises two disulfides that form a closed loop structure surrounding a third disulfide to form a rigid nut structure. The seventh cysteine contained in each monomer forms intramolecular disulfide bonds that covalently bond the monomers to form the final dimer product (Rattenholl et al 2000, J. Mol. Biol , 305: 523).

TGF beta family members are synthesized as pre-pro proteins that are secreted as mature homodimers after degradation of signal peptides and pro-domains (eg, Rattenholl, et al., 2000, J. Mol. Biol , 305: 523; See Fairlie et al., 2001, J. Biol Chem. , 276 (20): 16911). Both signal peptides and pro-domains modulate appropriate secretion for TGF beta family members (Rattenholl et al., 2000, J. Mol Biol , 305: 523; Rattenholl et al., 2001, Eur. J. Biochem. , 268: 3296).

Summary of the Invention

The present invention is based, at least in part, on the discovery that certain buffer compositions are particularly effective at inducing refolding of denatured polypeptides. The compositions and methods described in detail herein have been developed to induce protein refolding to produce polypeptides with appropriate three-dimensional structures and with biological activity.

In one embodiment, the present invention comprises the steps of (1) providing a modified polypeptide; And (2) contacting the polypeptide with an amount of refolding buffer effective to induce folding of the polypeptide, wherein the refolding buffer comprises: i) potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM at pH 5.8 to 8.0, (ii) guanidine-HCl at a concentration of 0.3 M to 2 M, (iii) L-arginine at a concentration of 0.25 M to 1 M, (iv ) Tween-80 at a concentration of 0.05% to 1% and (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, wherein the ratio of oxidized glutathione to reduced glutathione is 5: 1 to 20: 1.

In certain embodiments, the modified polypeptide is a polypeptide comprising a TGF beta superfamily member.

As used herein, a "TGF beta superfamily member" is a protein having the same sequence as the wild type member of the TGF beta superfamily, a cleavage that retains the biological activity of the wild type protein, or a wild type protein (full length or mature protein) It refers to a variant having at least 70% sequence identity for and retaining the biological activity of the wild type protein. Examples of members of the TGF beta superfamily include TGF-beta, growth differentiation factors, bone morphogenic proteins, activin, inhibin, and glial cell line-derived nerve growth factors. In certain embodiments, the variants have at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to the full length wild type protein and retain the biological activity of the wild type protein. In certain embodiments, the variants have at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to the mature wild type protein and retain the biological activity of the wild type protein.

The description of the concentration of the "refolding buffer" component used in the methods described herein refers to the final concentration of the refolding buffer component present in the reaction with the modified polypeptide (the refolding buffer prior to addition of the other components of the folding reaction). Not the concentration of the components in the stock solution.

As used herein, "to induce folding of a polypeptide" refers to the induction of the tertiary structure of a polypeptide corresponding to that of a wild-type protein, and the acquisition of related biological activities.

TGF beta superfamily members may be members of the glial cell line-derived neuronal growth factor (GDNF) family. As used herein, "GDNF family member" means a protein having the same sequence as a wild type member of the GDNF family, a cleavage that retains the biological activity of the wild type protein, or sequence identity to a wild type protein (full length or mature protein). More than 70% and retains the biological activity of the wild type protein. Members of the GDNF family include GDNF, neuroturin, nublastine and percepin. In certain embodiments, the variants have at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to the full length wild type protein and retain the biological activity of the wild type protein. In certain embodiments, the variants have at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to the mature wild type protein and retain the biological activity of the wild type protein.

In certain embodiments, the GDNF family member is a nublastin protein. As used herein, a "nublastine protein" refers to a protein having the same sequence as a wild type nublastin (eg, human nublastin), a cleavage that retains the biological activity of the wild type protein, or a wild type protein (whole Length or mature nublastin protein) to at least 70% sequence and retains the biological activity of the wild type protein. In certain embodiments, the variants have at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to the full length wild type protein and retain the biological activity of the wild type protein. In certain embodiments, the variant has at least 70%, 80%, 85%, 90%, 95% or 98% sequence identity to a mature wild type protein (eg, amino acid residues 108-220 of SEQ ID NO: 1) and is wild-type. Retains the biological activity of the protein. The nublastin protein may comprise or consist of, for example, amino acid residues 122-220 of SEQ ID NO: 1, amino acid residues 117-220 of SEQ ID NO: 1 or amino acid residues 108-220 of SEQ ID NO: 1.

Such methods may further comprise expressing the polypeptide in bacteria (eg, E. coli ) prior to inducing folding with the refolding buffer. In certain embodiments, the polypeptide is expressed in bacteria in insoluble form and the insoluble polypeptide is contacted with an amount of solubilization buffer effective to denature the polypeptide prior to inducing folding with the refolding buffer.

In certain embodiments, the refolding buffer comprises L-arginine at a concentration of 0.30 M to 0.5 M. In other embodiments, the refolding buffer comprises L-arginine at a concentration of at least 0.30 M. In other embodiments, the refolding buffer comprises L-arginine at a concentration of at least 0.35 M. In other embodiments, the refolding buffer comprises L-arginine at a concentration of 0.35 M.

In certain embodiments, the refolding buffer comprises Tween-80 at a concentration of 0.1% to 1%. In other embodiments, the refolding buffer comprises Tween-80 at a concentration of 0.1% to 0.5%. In other embodiments, the refolding buffer comprises Tween-80 at a concentration of at least 0.1%. In other embodiments, the refolding buffer comprises Tween-80 at a 0.1% concentration.

In certain embodiments, the refolding buffer comprises oxidized glutathione and reduced glutathione in a ratio of 5: 1 to 10: 1. In other embodiments, the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 ratio. In certain embodiments, the refolding buffer comprises oxidized glutathione at a concentration of 1 mM to 2 mM. In other embodiments, the refolding buffer comprises oxidized glutathione at a concentration of 1 mM.

In certain embodiments, the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M to 1.0 M. In other embodiments, the refolding buffer comprises guanidine-HCl at a concentration of at least 0.5 M. In other embodiments, the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M.

In certain embodiments, the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 100 mM. In other embodiments, the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 75 mM. In other embodiments, the refolding buffer comprises potassium phosphate at a concentration of at least 50 mM. In other embodiments, the refolding buffer comprises potassium phosphate at a concentration of 50 mM. In certain embodiments, the refolding buffer comprises potassium phosphate at pH 7.0 to 8.0. In other embodiments, the refolding buffer comprises potassium phosphate at pH 7.5 to 8.0.

In other embodiments, the refolding buffer comprises potassium phosphate at a pH of about 7.8. The refold buffer optionally comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-arginine at a concentration of 0.35 M, and (iv) Tween- at a concentration of 0.1%. 80, (v) oxidized glutathione at a concentration of 1 mM and (vi) reduced glutathione at a concentration of 0.2 mM.

In certain embodiments, the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-arginine at a concentration of 0.35 M, (iv) 0.1 Tween-80 at% concentration, (v) oxidized glutathione at 1 mM concentration and (vi) reduced glutathione at 0.2 mM concentration.

In certain embodiments, the refolding buffer lacks urea and / or glycine.

In another embodiment, the invention features a composition comprising an amount of refolding buffer effective to induce folding of the nublastin polypeptide when diluted 1 to 10 fold, wherein the refolding buffer comprises (i) pH Potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM from 5.8 to 8.0; (ii) guanidine-HCl at a concentration of 0.3 M to 2 M; (iii) L-arginine at a concentration of 0.25 M to 1 M; (iv) Tween-80 at a concentration of 0.05% to 1%; And (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, 1 to 10 times the concentration specified above, wherein the ratio of oxidized glutathione to reduced glutathione is 5 : 1 to 20: 1. Such compositions can optionally be used as stock solutions diluted with other components prior to initiating the folding reaction.

In certain embodiments, the refolding buffer comprises 1 to 10 times L-arginine at a concentration of 0.30 M to 0.5 M. In other embodiments, the refolding buffer comprises 1 to 10 times L-arginine at a concentration of at least 0.30 M. In other embodiments, the refolding buffer comprises 1 to 10 times L-arginine at a concentration of at least 0.35 M. In other embodiments, the refolding buffer comprises 1 to 10 times L-arginine at a concentration of 0.35 M.

In certain embodiments, the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of 0.1% to 1%. In other embodiments, the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of 0.1% to 0.5%. In other embodiments, the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of at least 0.1%. In other embodiments, the refolding buffer comprises 1 to 10 times Tween-80 at a 0.1% concentration.

In certain embodiments, the refolding buffer comprises oxidized glutathione and reduced glutathione in a ratio of 5: 1 to 10: 1. In other embodiments, the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 ratio. In certain embodiments, the refolding buffer comprises 1 to 10 times oxidized glutathione at a concentration of 1 mM to 2 mM. In other embodiments, the refolding buffer comprises 1 to 10 times oxidized glutathione at a concentration of 1 mM.

In certain embodiments, the refolding buffer comprises 1-10 times guanidine-HCl at a concentration of 0.5 M to 1.0 M. In other embodiments, the refolding buffer comprises 1-10 times guanidine-HCl at a concentration of at least 0.5 M. In other embodiments, the refolding buffer comprises 1-10 times guanidine-HCl at a 0.5 M concentration.

In certain embodiments, the refolding buffer comprises 1-10 times potassium phosphate at a concentration of 25 mM to 100 mM. In other embodiments, the refolding buffer comprises 1-10 times potassium phosphate at a concentration of 25 mM to 75 mM. In other embodiments, the refolding buffer comprises 1-10 times potassium phosphate at a concentration of at least 50 mM. In other embodiments, the refolding buffer comprises 1-10 times potassium phosphate at a 50 mM concentration. In certain embodiments, the refolding buffer comprises potassium phosphate at pH 7.0 to 8.0. In other embodiments, the refolding buffer comprises potassium phosphate at pH 7.5 to 8.0. In other embodiments, the refolding buffer comprises potassium phosphate at a pH of about 7.8.

The refold buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8; (ii) guanidine-HCl at a concentration of 0.5 M; (iii) L-arginine at a concentration of 0.35 M; (iv) Tween-80 at 0.1% concentration; (v) oxidized glutathione at 1 mM concentration; And (vi) optionally reduced glutathione at a concentration of 0.2 mM, from 1 to 10 times the stated concentration, or may consist of these components.

In certain embodiments, the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-arginine at a concentration of 0.35 M, (iv) 0.1 Tween-80 at% concentration, (v) oxidized glutathione at 1 mM concentration and (vi) reduced glutathione at 0.2 mM concentration.

In certain embodiments, the refolding buffer lacks urea and / or glycine.

The compositions and methods described herein adequately refold a large amount of TGF beta superfamily protein, such as nublastin, in an environment that is produced in a host (eg, a bacterium) in which the protein is not produced with a properly folded biologically active product. It is advantageous in that it can be purified and properly refolded protein.

Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although used to practice or test the present invention using methods and materials similar or equivalent to those described herein, the following describes methods and materials. All publications, patent applications, patents, and other cited references mentioned herein are incorporated herein by reference. In case of dispute, the present application, including definitions, controls. The materials, methods, and examples are presented by way of example only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

The present invention provides compositions and methods for inducing folding of denatured polypeptides belonging to the TGF beta superfamily. The application of certain compositions to induce folding of denatured nublastins that are members of the TGF beta superfamily and the GDNF subfamily is described in the Examples below. Since nublastin has a cysteine nut structure common to members of the TGF beta superfamily and the GDNF subfamily, the refolding buffers described herein induce folding of other polypeptides belonging to the TGF beta superfamily and GDNF subfamily. It is expected to be effective.

New blastine

Natural human pre-pro New vinblastine polypeptide is 220 amino acids in length and has a sequence of to: M A S ELGLGGLSTLSHCPWPRRQPALWPTLAALALLSSVAE LGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSGRAREPPPQPSRPAPPPPAPPSALPRGGRAA AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNST WRTVDRLSATACGCLG R (SEQ ID NO: 1).

Human nublastin signal peptide starts with methionine (underlined) at position 1 and ends with alanine (underlined) at position 39. The full length prodomain of human nublastine begins with serine (underlined) at position 40 and ends with arginine (underlined) at position 107. The mature human nublastin polypeptide consists of the carboxy terminal 113 amino acid starting with alanine at position 108 and ending with glycine at position 220. The compositions and methods described herein can be used for the preparation of denatured nublastin proteins, including full-length nublastins, mature nublastins (signal peptide and prodomain deficiency), or biologically active cleavage or variants of mature nublastins. Provide effective folding

Nublastin proteins folded by the methods described herein can vary in length. Mature human nublastin polypeptides consist of the 113 amino acids at the carboxy terminus of prepronublastin, but not all 113 amino acids are required to obtain useful nublastin biological activity. Amino terminal truncation is acceptable. Thus, the new blastin polypeptide is a carboxy terminal 99-113 amino acid of natural human nublastin (ie, its length is 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, May be 112 or 113 amino acids). Nublastine polypeptides consisting of the carboxy terminus 104 and 113 amino acids of nublastine are exemplified in the Examples presented below.

In addition to varying lengths, the nublastin polypeptides may vary in sequence. In particular, certain amino acid substitutions may be introduced into the nublastin sequence without significantly losing the nublastin biological activity described herein. In an exemplary embodiment, the polypeptide has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO 1 (or identity to amino acids 108-220 of SEQ ID NO 1). 70%, 80%, 85%, 90%, 95%, 98% or 99%). Variant nublastin polypeptides differing in sequence from those disclosed in SEQ ID NO: 1 (or amino acids 108-220 of SEQ ID NO: 1) may comprise one or more conservative amino acid substitutions, one or more non-conservative amino acid substitutions, and / or one or more deletions or insertions. It may include. In certain embodiments, variant nublastin polypeptides comprise one or more amino acid substitutions to SEQ ID NO: 1 (or amino acids 108-220 of SEQ ID NO: 1), which are polymers (eg, polyalkylene glycol moieties such as polyethylene glycol) Moieties) to provide internal polymer conjugation sites (an example neublastin variant is disclosed in WO02 / 060929, which is incorporated herein by reference). In certain embodiments, variant nublastin polypeptides comprise one or more amino acid substitutions (eg, non-conservative substitutions) for SEQ ID NO: 1 (or amino acids 108-220 of SEQ ID NO: 1), which means heparin binding (eg, R155E, R156E, R158E or R155,156E or at least one of these substitutions at that position or positions in the mature nublastin polypeptide).

Conservative substitutions typically include valine, alanine and glycine; Leucine, valine and isoleucine; Aspartic acid and glutamic acid; Asparagine and glutamine; Serine, cysteine and threonine; Lysine and arginine; And substituting one amino acid for other amino acids having similar characteristics, such as substitution in groups of phenylalanine and tyrosine. Examples of polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Examples of polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Examples of positively charged (basic) amino acids include arginine, lysine and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the aforementioned polar, basic or acidic group with another member of the same group may be considered conservative substitution.

The polypeptides used in the methods described herein may comprise heterologous amino acid sequences in addition to nublastin proteins. As used herein, the term “heterologous” as used herein refers to a sequence derived from a foreign source for a particular host cell or modified from its initial form when from the same host cell. Examples of heterologous sequences include heterologous signal sequences (eg, native rat albumin signal sequences, modified rat signal sequences, or human growth hormone signal sequences) or sequences used for purification of nublastin proteins (eg histidine labels).

Nublastin activity

Nublastin polypeptides used in the methods described herein exhibit one or more biological activities of natural nublastin. Biologically active nublastin polypeptides are polypeptides that, when dimerized, can bind to RET with GFRα3 and induce RET dimerization and autophosphorylation (eg, Sanicola et al., 1997, Proc . Natl. Acad) . . ScL USA , 94: 6238). Any method of determining receptor binding and receptor autophosphorylation can be used to assess the biological activity of the nublastin polypeptide. For example, the KIRA assay described in Example 3 can be used to assess nublastin biological activity (see also Sadick et al., 1996, Anal . Biochem ., 235 (2): 207).

Refold buffer

In general, the refolding buffer used in the methods described herein comprises (i) potassium phosphate at a concentration of 25 mM to 150 mM; (ii) guanidine-HCl at a concentration of 0.3 M to 2 M; (iii) L-arginine at a concentration of 0.25 M to 1 M; (iv) Tween-80 at a concentration of 0.05% to 1%; And (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, wherein the ratio of oxidized glutathione to reduced glutathione is 5: 1 to 20: 1.

In certain embodiments, sodium phosphate at a concentration of 25 mM to 150 mM may be used in place of potassium phosphate. The pH of sodium phosphate or potassium phosphate used in this process is generally between 5.8 and 8.0. Also, in certain embodiments, detergents such as Tween-20 or NP40 may be used in place of Tween-80 at a concentration of 0.05% to 1%.

The effect of certain refolding buffers on inducing folding of denatured polypeptides can be assessed by measuring absorbance (OD 320) after incubation of denatured polypeptides in specific buffers (Example 1 and FIG. 2). The absorbance measured in this assay indicates the presence of precipitated and improperly folded protein. As shown in Example 2 of the Examples below, low absorbance readings indicate that the buffer is effective for inducing folding of the denatured polypeptide. In addition, the biological activity of the folded polypeptide can be measured by the in vitro and / or in vivo biological assays described herein.

The following is an example of the practice of the present invention. It should not be construed as limiting the scope of the invention in any way.

1 depicts the sequences of human and rat 113 amino acids and 104 amino acid forms of nublastin.

FIG. 2 is a graph depicting the absorbance detected after incubation of solubilized nublastin of refolding buffer (buffer 4 with 1% concentration of Tween-80, not shown) detailed in Table 1. FIG. to be.

Example  One: New blastine Refolding Buffer  Confirm

Recombinant nublastin was expressed as 10 histidine-labeled fusion protein (FIG. 1) in E. coli under the control of the T7 promoter. Both human and rat 113 and 104 amino acid forms are derived from their respective constructs (FIG. 1) and refolded and purified by the methods described herein. In FIG. 1, the start of 113 amino acid and 104 amino acid forms is underlined and shown in bold.

When expressed in E. coli , nublastine is included as an insoluble protein in inclusion bodies (IB). Therefore, to obtain a soluble and bioactive product, nublastin must be separated from the IB and refolded. Inclusion bodies were obtained by lysing Escherichia coli ( E. coli ) expressing New Blastin in PBS using a Gaulin press, followed by centrifugation. Unless otherwise noted, all centrifugations were performed at 4 ° C. and all other steps were performed at room temperature. In order to obtain properly refolded nublastin in the maximum yield possible, it is advantageous to start from IB pellets free of cell debris. To accomplish this, the IB pellets were weighed and further washed in IB wash buffer (20 mM Tris pH 8.5 and 0.5 M EDTA; 8 mL per g of protein). IB pellets were collected by centrifugation at 15,000 × g for 20 minutes, discarded the opaque supernatant and washed again in the same buffer (8 ml per g of protein) containing 2% Triton-X 100 to remove contaminating lipids. . Final washing was performed to remove Tween-80 using detergent-free wash buffer (8 ml per g of protein) and discard the supernatant again.

Freshly made solubilization buffer (6M guanidine-HCl, 0.05 M potassium phosphate pH 7.8, 0.1 M DTT and 1.0 mM EDTA) was added to the pellet and mixed well using a Polytron mixer. To confirm that solubilization was complete, the mixture was stirred at rt overnight. The next day, the solution became clear by centrifugation at 10,000 rpm for 20 minutes. The supernatant was transferred to a new container and the remaining insoluble pellets were weighed to assess the recovery. Not all proteins were solubilized by this method. Soluble proteins were quantified using standard Bradford protein assays using BSA solubilized as a control.

A potent series of refolding buffers were prepared in 96 well plates to determine if specific buffer conditions could yield adequately refolded nublastine in high yield (see Table 1).

As shown in Table 1 above, guanidine HCl (0.5 M), reduced glutathione (0.2 mM) and potassium phosphate pH 7.8 (50 mM) were kept constant on the plate while L-arginine, oxidized glutathione and Tween The concentration of -80 changed. L-arginine varied from 0.15 M to 0.35 M (same addition of less than 0.8 M L-arginine was performed) and oxidized glutathione was changed from 1 mM to 4 mM. In addition, Tween-80 was changed from 0% to 1%. Since glycine in certain cases may replace L-arginine during refolding, a separate plate was prepared that kept all buffer components identical except for replacing L-arginine with 25-100 mM glycine. It was.

The final volume of buffer in each well was 280 μl (freshly reduced glutathione from stock concentrate was added). 20 μl of solubilized nublastin was then added to each well at a concentration of 0.1 mg / ml. Absorbance was monitored over a 48 hour period. Any detected absorbance indicates the presence of a protein that is precipitated and not properly refolded.

Most precipitation occurred in wells containing 0.15 M L-arginine and minimal precipitation was observed in wells containing 0.35 M L-arginine (FIG. 2). Of the wells containing 0.35 M L-arginine, the best overall results were observed in the wells containing 0.1% Tween-80. Best refolding was observed when the ratio of oxidized glutathione to reduced glutathione was 20: 1 (however, for the further experiments described herein to reduce the content of oxidized glutathione required for the refold buffer) 1: 1 ratio). Based on this criterion, the refolding buffer system shown in the examples below was used, providing high yield and properly refolded nublastin. Under all buffering conditions, the use of glycine instead of L-arginine resulted in nublastin precipitation.

Example 2: Refolding and Purification of Nublastine

0.5 M guanidine-HCl, 0.35 M L-arginine, 50 mM potassium phosphate pH 7.8, 0.2 mM reduced glutathione, 1 mM oxidation using the results of the buffer assay described in Example 1 applied in this example and the following examples Prepared refolding buffer of glutathione and 0.1% Tween-80. Refold buffer was prepared fresh. Solubilized protein was rapidly diluted with refolding buffer to a final protein concentration of 0.05-0.5 mg / ml. On average, 0.1 mg / ml of solubilized nublastin was used. This mixture was incubated for at least 48 hours at room temperature. Stirring was not necessary.

Host Cell Contaminant Removal Using Ni-IMAC Chromatography

L-arginine was diluted from 0.35 M to 0.175 M to prevent leaching of Ni from IMAC resin. This can be done using one of the following methods. Arginine can be diluted directly to the appropriate concentration with 0.5 M guanidine-HCl. Water was not used alone, which would lead to significant loss in product recovery if precipitation of nublastine resulted in the absence of guanidine concentration (guanidine-HCl must be maintained in the buffer until the cation chromatography step described below). Because. Since direct dilution of L-arginine substantially increased the working volume and increased the amount of guanidine required, the protein was concentrated to 1/20 times the initial volume using the Millipore tangential flow Pellicon unit. After concentration, L-arginine was diluted to 0.175 M using 0.5 M guanidine.

The diluted L-arginine solution was added to a Ni-NTA IMAC column previously equilibrated in column wash buffer (40 mM imidazole and 0.5 M guanidine HCl) using a flow rate of 50-100 ml per minute. Nublastine binds to the Ni-NTA matrix via histidine labeling, and no product was observed in the flow through. After washing with 5 column volumes of wash buffer, nublastine was eluted from the resin using 0.2 M imidazole in 0.5 M guanidine. The column wash buffer (without neublastine) was discarded. Bradford assay was used to monitor protein recovery. In addition, host cell contaminants were monitored from this point onward.

Histidine Labeling Isolation from Nublastin by Protease Digestion

One of two possible histidine labeling methods was used, depending on the length of nucleostin required (113 amino acids or 104 amino acids).

To generate the wild type 113 amino acid nublastin product, Endo Lys C was used to clip the label c-terminus of the lysine residue contained in the label. Five units of Endo Lys C (WAKO, Catalog # 129-02541) per gram of nublastine were added to the material from the Ni-NTA eluate. No buffer replacement or pH adjustment was necessary (in certain cases buffers with 10 mM Hepes pH 7.8 were used instead and worked effectively). Nublastin with protease was incubated overnight with constant stirring at room temperature.

To produce 104 amino acid forms from nublastin, histidine-labeled products were treated with trypsin (Cooper Biomedical # 3740) using a ratio of trypsin: nublastin of 1: 2,000. No buffer replacement or pH adjustment is necessary. The mixture was incubated overnight with constant stirring at room temperature.

Ni-NTA resin was equilibrated with buffer (0.5 M guanidine-HCl and 0.04 M imidazole). After adjusting the imidazole concentration in the nublastin formulation from 0.2 M to 0.04 M using 0.5 M guanidine-HCl, the material was added to the Ni-NTA resin at a flow rate of 50 to 100 ml per minute. Column passages containing non-labeled New Blastin were collected and monitored for New Blastin using Bradford assay. To regenerate the Ni-NTA resin, the histidine label was eluted using 0.2 M imidazole in 0.5 M guanidine HCl. This material was treated with SDS / PAGE together with the resin flow through to confirm the efficiency of protease digestion.

The Ni-NTA flow-through from this step was adjusted to 0.35 M guanidine-HCl by adding ddH ₂ O. High concentrations of guanidine can prevent nublastin from binding to the cationic substrate. The C-100 filter binding cartridge (Sartorious, Catalog # ClOOX) was equilibrated with C-100 wash buffer (5 mM sodium phosphate pH 6.5 and 0.35 M NaCl).

SP-Sepharose (Amersham Pharmacia) can be used in place of the C-100 membrane filter. However, C-100 was chosen because of its larger surface area compared to conventional column chromatography. Purification of nublastine on SP-Sepharose may prevent the local aggregation of nublastine by selecting a larger column diameter and / or reducing protein loading. This prevents local high concentrations of nublastin, which may contribute to tetramer formation and product precipitation, especially when using sodium phosphate buffer.

Nublastine in 0.35 M guanidine-HCl was added to the C-100 filter at a flow rate of 50 to 100 ml per minute, and then the filter was rinsed strongly with C-100 washing buffer. This step removes any remaining histidine label, endotoxin and nublastin monomer. Nublastine dimers were recovered by eluting the protein from the C-10 matrix using 5 mM sodium phosphate pH 6.5 and 1 M sodium chloride. Elution was monitored by UV absorbance at 280 nm and Newblastin peak collected in one vessel.

New blastine  Thickening and Buffer  how

Nublastine was concentrated by Millipore Biomax-10 tangential flow filtration and diafiltered with 5 mM sodium phosphate pH 6.5 and 0.15 M sodium chloride with 5 diafiltration volumes using the same units. Attempts were made to obtain 1.0-1.5 mg / ml final protein concentrations and concentrations were not higher than 2.0 mg / ml, otherwise nublastin could initiate precipitation in the formulation with significant protein loss. Once the product was concentrated to 1.0 mg / ml and combined in 5 mM sodium phosphate pH 6.5 and 0.15 M sodium chloride, nublastine was divided into convenient portions and stored at -70 ° C until needed.

Example 3: Analytical Characterization of Nublastin

The purified nublastin described in Example 2 was subjected to various assay tests to demonstrate purity, primary amino acid sequence, live activity and disulfide structure integrity.

SDS / PAGE evaluation of purity and molecular weight

Samples from each nublastin refolding / purification step were subjected to SDS / PAGE analysis through 4-20% acrylamide gels under non-reducing conditions. The final nublastin product migrated as a reducing dimer of 24,000 Da and the purity evaluated was> 98%.

Mass Spectrum Analysis of Refolded Rat New Blastin

In order to assess the purity of the refolded product and to determine the mass, nublastine was subjected to mass spectral analysis on a ZMD mass spectrometer. Nublastine was denatured in 8 M urea and treated with DTT prior to analysis to reduce all disulfide bonds and convert dimer molecules to monomers. The main signal identified indicates rat nublastin residues 10-113, suggesting that it is the predominant type in the formulation as expected. However, a major signal at 10991 Da was identified and expected to correspond to a leucine deletion, and the signal at 11076 is expected to be a small amount of arginine for lysine substitution. Low levels of peaks correspond to oxidation, acetonitrile adducts and TFA adducts. In addition, a small amount of 106 amino acid form of nublastin was identified. Trypsin-related peaks were not identified.

Characterization of Rat 104 Amino Acid Nublastin by AspN Peptide Mapping

AspN peptide mapping was performed on nublastin produced by trypsin digestion to remove histidine labels. This batch was compared to a number of other nublastin preparations, including wild type rat 113 amino acids, wild type human 113 amino acids, human 104 amino acid forms. These results indicated that this batch exemplified about 8% oxidation in Met92, 5% Leu61 deletion, a low degree of Arg to Lys transition and less than 1% deamidation in Asn95, as expected.

Disulfide Assay of Rat 104 Amino Acid Nublastin

Disulfide analysis was performed on rat 104 amino acid nublastin. Wild-type rat 113 amino acid nublastin was simultaneously performed as a reference example. About 150 μl of refolded and purified nublastin was used for disulfide mapping. The results indicate that all disulfide bonds in the two samples are similar and as expected. The profile of the nublastin monomer is similar to the reference example except for the area of low degree peak eluted directly above the major monomer peak. This initial elution peak is expected to include partially oxidized monomers and is not included in the downstream weight mapping. Fractions containing disulfide-bound peptides were pooled and analyzed by MALDI-TOF mass spectrum using DHB as a metric. According to the data, rat 104 amino acid nublastin after AspN / trypsin digestion was as expected, and there is no evidence of mixed disulfide connectivity.

Analysis of Nublastin Activity Using Kinase Receptor Activation-Enzyme-linked Immunosorbent Assay

Nublastin activity was measured through the ability to stimulate c-Ret phosphorylation in NB41A3-mRL3 cells, an adherent murine neuroblastoma cell line expressing Ret and GFRα3. NB41A3-mRL3 cells were cultured in a 5% CO ₂ at 2 × 10 ⁵ per well inoculated with 10% FBS to a cell in a supplemented DMEM, and 37 ℃ for 18 hours in a 24-well plate. After removing the medium and washing the cells with 1 ml PBS per well, the cells were stimulated with DMEM containing 113 amino acids or 104 amino acids nublastin for 10 minutes at 37 ° C. and 5% CO ₂ . To stop nublastin activity, remove the medium, and immediately before lysis using 10 mM Tris, pH 8.0, 0.5% NP40, 0.2% DOC, 50 mM NaF, 0.1 mM Na ₃ VO ₄ and 1 mM PMSF. Was washed with PBS. After 1 hour incubation at 4 ° C, lysates were mixed with repeated pipetting and transferred to 96-well ELISA plates coated with anti-RET mAb (AA.GE7.3) (0.25 ml per well). Wells were blocked with blocking buffer (TBST with 1% normal mouse serum and 3% BSA) for 1 hour at room temperature and then washed 6 times with TBST alone. The captured receptor was incubated with HRP-conjugated phosphotyrosine antibody (4G10; 0.2 μg of welding) (2 hours) to detect phosphorylated RET. After incubation, the wells were washed 6 times with TBST and HRP activity was detected at 450 nm by colorimetric analysis. Absorbance values from wells treated with lysate or lysate buffer alone were measured, background corrected, and the data plotted as a function of the concentration of nublastine present in the activation mixture. Rat 104 amino acid nublastin was active in the KIRA assay as in the positive 113 amino acid nublastin control, meaning that the refolding / purification process yields a biologically active product.

Endotoxin analysis

Endotoxin levels in each of the purification steps were measured using Limulus cell line lysate assay and manufacturer's suggested conditions (Bio * Fecker). Most endotoxins were removed in the first Ni-NTA cleaning step. After trypsin addition, endotoxin levels were observed to increase slightly due to endotoxin in most used trypsin formulations. Cleaning the C100 column with a large amount of wash buffer appears to be useful in removing residual endotoxins. The endotoxin level in the final product was below the maximum allowable level.

Host Cell Protein Analysis

Host cell protein contamination was monitored at each purification step using the E. coli host cell protein assay kit from Cygnus Technologies and the conditions suggested by the manufacturer. This kit is an ELISA based assay with sensitivity up to 1 ng / ml host cell protein. Using the endotoxin results above, removal of most host proteins occurred during primary Ni-NTA chromatography as well as during C100 washing. Host cell protein was determined to be less than 0.0001% of the final product.

Trypsin removal assay

Trypsin removal was monitored using fluorometer analysis using N-T-BOC-GLN-ALA-ARG 7-AMIDO-4-methylcoumarin HCl as substrate and sensitivity was less than 40 ng / ml. Most, but not all, of the added trypsin was removed from the C100 effluent by washing. The amount of trypsin remaining in the final product was less than 0.004% (below the sensitivity level).

Histidine-labeled detection ELISA

Histidine labeled ELISAs using anti-polyhistidine antibodies have been developed to monitor histidine-labeled nublastin remaining in the final formulation. As expected, most histidine labels were found in the material prior to the first Ni-NTA, but not in the first Ni-NTA flow-through fluid, indicating that most histidine-labeled nublastins were Ni-NTA resins. It is bound to. This material was eluted from the resin with a 0.2 M imidazole elution. The sensitivity of this assay was about 0.3 μg / ml and the final content of histidine-labeled nublastin found in the final product was determined to be 0.12% or 0.88 mg of total protein.

Host Cell DNA Detection Assay

The removal rate of host cell DNA was monitored by an assay using a single stranded DNA binding protein bound to avidin in a sandwich assay based on ELISA. This analysis demonstrated a sensitivity up to about 200 pg / ml of E. coli DNA. Based on the single stranded DNA binding assay, the final nublastin preparation was determined to have less than 0.0001% contaminating host cell DNA. As with the other assays described above, both the first Ni-NTA chromatography step and the C100 wash step were most effective in removing DNA impurities in the starting material.

Chronic Contractile Injury (CCI) Rats Treated with 104 Amino Acid Nublastin

Nublastine-treated CCI rats were shown to have reduced tactile allodynia compared to vehicle-treated controls. Rats treated with New Blastin were able to withstand the greater force applied to ipsilateral paws. Tactile allodynia was evaluated using von Frey Hairs applying the up-down method (Chaplan et al., 1994). Rats were tested at days 7, 10, 14, 17 and 21 for modified pain thresholds. Siamese (n = 3) did not show different gram thresholds during the test period, but all CCI rats had a lower limit for von Frey Hairs applied relative to their baseline values. Rats treated with Newblastin-104 1 mg / kg (n = 8) and 3 mg / kg (n = 7) were able to withstand elevated limits compared to vehicle treated controls (n = 8). The tolerability of the nublastin treated animals has statistical significance (p <.05) in CCI after 17 and 21 days of surgery. Heat hyperalgesia was attenuated in Newblastine treated CCI rats, with the 3 mg / kg dose showing higher efficiency than the 1 mg / kg dose at 21 days postoperatively. Heat hyperalgesia was measured using a Hargreaves device to assess the heat shrink latency. Rats were tested at days 7, 10, 14, 17 and 21 for reduced limb atrophy latency. Siamese (n = 3) did not show modified limb atrophy latency during the test period, but all CCI rats have a short limb atrophy latency than their baseline value. Nublastin-104 1 mg / kg (n = 8) and 3 mg / kg (n = 7) resulted in longer heat stimulation applications compared to the vehicle control (n = 8) at 14, 16 and 21 days after CCI induction. Can withstand The 104 amino acid nublastin 3 mg / kg-treated rats showed a significantly higher incubation period 21 days post-operatively compared to the 104 amino acid nublastine 1 mg / kg treated rats, Limb atrophy incubation period had statistical significance (p <.05) at 14, 17 and 21 days after CCI surgery.

CCI rats treated with New Blastin can add more weight to the diseased acute atrophy of the lower limb, as can be seen using the incapacitance test. In-capacitance was measured using an in-capacitance meter to assess the weight distribution of each foot. At baseline, the rats distributed the same weight between the feet, but after injury the smaller body weights were added to the ipsilateral paws. Siamese (n = 4) did not show a modified weight dispersion between the feet during the test period, but all CCI rats weighed less on the affected foot compared to their baseline values. The 104 amino acid nublastin 1 mg / kg (n = 8) and 3 mg / kg (n = 7) added more weight to the ipsilateral paw compared to the vehicle treated control (n = 8). Incapacitance of diseased paws in New Blastin treated animals has statistical significance (p <.05) at 14, 17 and 21 days after CCI surgery.

There is no statistically significant difference between nublastin and vehicle treated CCI rats in the cold allodynia test, but nublastin-treated rats tend to have a shorter duration at 10 days. Low temperature allodynia was measured for 5 minutes using a copper cold plate cooled to 4 ° C. Rats were tested at days 7, 10, 14, 17 and 21 with increased limb atrophy periods relative to their baseline values. At baseline no animals responded to low temperatures. Siamese (n = 3) do not exhibit increased limb atrophy duration during the test period, but all CCI rats have increased limb atrophy duration relative to their baseline values. Although the time of limb atrophy by nublastin treated animals was not statistically significant, 104 amino acids nublastin 1 mg / kg (n = 8) and 3 mg / kg (n = 7) were found after CCI induction. On days 17 and 21, the affected limbs increased for a shorter time compared to the vehicle treatment group (n = 8).

Other embodiments

Although the present invention has been described in detail with reference to the description, the above description is intended to illustrate rather than limit the scope of the invention, the scope of the invention is defined by the appended claims below. Other embodiments, advantages, and modifications are included in the following claims.

Claims (64)

Providing a denatured polypeptide comprising a transforming growth factor beta (TGF beta) superfamily member; And Contacting said polypeptide with an amount of refolding buffer effective to induce folding of said polypeptide, The refolding buffer comprises (i) potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM at pH 5.8 to 8.0, (ii) guanidine-HCl at a concentration of 0.3 M to 2 M, (iii) a concentration of 0.25 M to 1 M L-arginine, (iv) Tween-80 at a concentration of 0.05% to 1% and (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, wherein the oxidized glutathione And a ratio of reduced glutathione to 5: 1 to 20: 1. The method of claim 1, wherein the refolding buffer comprises L-arginine at a concentration of 0.30 M to 0.5 M. 3. The method of claim 1, wherein the refolding buffer comprises L-arginine at a concentration of 0.30 M or greater. The method of claim 1, wherein the refolding buffer comprises L-arginine at a concentration of 0.35 M or greater. The method of claim 1, wherein said refolding buffer comprises 0.35 M concentration of L-arginine. 6. The method of claim 1, wherein the refolding buffer comprises Tween-80 at a concentration of 0.1% to 1%. 7. The method of claim 1, wherein the refolding buffer comprises Tween-80 at a concentration of 0.1% to 0.5%. 6. The method of claim 1, wherein the refolding buffer comprises Tween-80 at a concentration of at least 0.1%. 7. 6. The method of claim 1, wherein the refolding buffer comprises Tween-80 at a concentration of 0.1%. 7. The method of claim 1, wherein the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 to 10: 1 ratio. The method of claim 1, wherein the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 ratio. 6. The method of claim 1, wherein the refolding buffer comprises oxidized glutathione at a concentration of 1 mM to 2 mM. 7. The method of claim 1, wherein the refolding buffer comprises oxidized glutathione at a concentration of 1 mM. 6. The method of claim 1, wherein the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M to 1.0 M. 7. 6. The method of claim 1, wherein the refolding buffer comprises guanidine-HCl at a concentration of at least 0.5 M. 7. 6. The method of claim 1, wherein the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M. 7. The method of any one of claims 1 to 5, wherein the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 100 mM. The method of any one of claims 1 to 5, wherein the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 75 mM. 6. The method of claim 1, wherein the refolding buffer comprises potassium phosphate at a concentration of at least 50 mM. 7. 6. The method of claim 1, wherein the refolding buffer comprises potassium phosphate at a concentration of 50 mM. 7. 6. The method of claim 1, wherein the refolding buffer comprises potassium phosphate at pH 7.0 to 8.0. 7. 6. The method of claim 1, wherein the refolding buffer comprises potassium phosphate at pH 7.5 to 8.0. 7. 6. The method of claim 1, wherein the refolding buffer comprises potassium phosphate at a pH of about 7.8. 7. The method according to any one of claims 1 to 5, wherein the refolding buffer comprises (i) 50 mM potassium phosphate at pH 7.8, (ii) guanidine-HCl at 0.5 M concentration, and (iii) 0.35 M concentration. L-arginine, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM. The method of claim 1 wherein the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-arginine at a concentration of 0.35 M, (iv) 0.1 Tween-80 at a concentration of%, (v) oxidized glutathione at a concentration of 1 mM and (vi) reduced glutathione at a concentration of 0.2 mM. The method of claim 1 wherein the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-arginine at a concentration of 0.35 M, (iv) 0.1 Tween-80 at% concentration, (v) oxidized glutathione at 1 mM concentration and (vi) reduced glutathione at 0.2 mM concentration. The method of claim 1, wherein the refolding buffer lacks urea. The method of claim 1, wherein the refolding buffer lacks glycine. 6. The method of claim 1, wherein the TGF beta superfamily member is a glioma cell line-derived neuronal growth factor (GDNF) family member. 7. The method of claim 29, wherein said GDNF family member is a nublastin protein. 31. The method of claim 30, wherein the nublastin protein comprises amino acid residues 122-220 of SEQ ID NO: 1. 31. The method of claim 30, wherein said nublastin protein comprises amino acid residues 117-220 of SEQ ID NO: 1. 32. The method of claim 30, wherein said nublastin protein comprises amino acid residues 108-220 of SEQ ID NO: 1. 27. The method of any one of claims 1-5, 25 or 26, further comprising expressing the polypeptide in bacteria prior to inducing folding into the refolding buffer. The method of claim 34, wherein the bacterium is E. coli . 35. The method of claim 34, wherein said polypeptide is expressed in bacteria in an insoluble form and contacting said insoluble polypeptide with an amount of solubilization buffer effective to denature the polypeptide prior to inducing folding with a refolding buffer. A composition comprising an amount of refolding buffer effective to induce folding of a nublastin polypeptide upon dilution at a rate of 1 to 10, wherein the refolding buffer comprises (i) a 25 mM to 150 mM concentration of pH 5.8 to 8.0. Potassium phosphate or sodium phosphate; (ii) guanidine-HCl at a concentration of 0.3 M to 2 M; (iii) L-arginine at a concentration of 0.25 M to 1 M; (iv) Tween-80 at a concentration of 0.05% to 1%; And (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, 1 to 10 times the concentration specified above, wherein the ratio of oxidized glutathione to reduced glutathione is 5: 1 to 20: 1. The composition of claim 37, wherein the refolding buffer comprises 1 to 10 times L-arginine at a concentration of 0.30 M to 0.5 M. 38. 38. The composition of claim 37, wherein the refolding buffer comprises 1 to 10 times L-arginine at a concentration of at least 0.30 M. 38. The composition of claim 37, wherein the refolding buffer comprises 1 to 10 times L-arginine at a concentration of at least 0.35 M. 38. The composition of claim 37, wherein the refolding buffer comprises 1 to 10 times L-arginine at a concentration of 0.35 M. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of 0.1% to 1%. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of 0.1% to 0.5%. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of at least 0.1%. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1 to 10 times Tween-80 at a concentration of 0.1%. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 to 10: 1 ratio. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises oxidized glutathione and reduced glutathione in a 5: 1 ratio. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times oxidized glutathione at a concentration of 1 mM to 2 mM. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises oxidized glutathione 1 to 10 times the 1 mM concentration. 42. The composition of any one of claims 37-41, wherein said refolding buffer comprises 1-10 times guanidine-HCl at a concentration of 0.5 M to 1.0 M. 42. The composition of any one of claims 37-41, wherein said refolding buffer comprises guanidine-HCl at a concentration of at least 0.5 M. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times guanidine-HCl at a concentration of 0.5 M. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times potassium phosphate at a concentration of 25 mM to 100 mM. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times potassium phosphate at a concentration of 25 mM to 75 mM. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times potassium phosphate at a concentration of at least 50 mM. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises 1-10 times potassium phosphate at a concentration of 50 mM. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises potassium phosphate at pH 7.0 to 8.0. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises potassium phosphate at pH 7.5 to 8.0. 42. The composition of any one of claims 37-41, wherein the refolding buffer comprises potassium phosphate at a pH of about 7.8. 42. The method of any one of claims 37-41, wherein the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) 0.35 M concentration. L-arginine, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM. The method of claim 37, wherein the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8; (ii) guanidine-HCl at a concentration of 0.5 M; (iii) L-arginine at a concentration of 0.35 M; (iv) Tween-80 at 0.1% concentration; (v) oxidized glutathione at 1 mM concentration; And (vi) reduced glutathione at a concentration of 0.2 mM, from 1 to 10 times the concentrations indicated above. The method of claim 37, wherein the refolding buffer comprises (i) potassium phosphate at a concentration of 50 mM at pH 7.8; (ii) guanidine-HCl at a concentration of 0.5 M; (iii) L-arginine at a concentration of 0.35 M; (iv) Tween-80 at 0.1% concentration; (v) oxidized glutathione at 1 mM concentration; And (vi) a reduced glutathione at a concentration of 0.2 mM, consisting of 1 to 10 times the concentration specified above. 42. The composition of any one of claims 37-41, wherein the refolding buffer lacks urea. 42. The composition of any one of claims 37-41, wherein said refolding buffer is deficient in glycine.

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